U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service

Veterinary Services Digitally signed by ROSEMARY ROSEMARY SIFFORD 

VS Guidance 13407.3 Date: 2023.08.21 18:05:56 SIFFORD -04'00' 

Tes�ng of Equids During Import Quarantine

1. Purpose and Background

This document establishes guidelines for tes�ng quaran�ned equids to determine their

import eligibility. These tes�ng requirements apply to all equids, including horses,

donkeys, mules, asses, and zebras. This guidance uses the terms “equid” and “horse”

interchangeably.

Veterinary Services (VS) personnel test horses presented for import to exclude four

diseases from the United States: dourine, glanders, equine infec�ous anemia (EIA), and

equine piroplasmosis (EP). Horses must test nega�ve on official tests to these four

diseases before VS will release them from quaran�ne and allow entry into the United

States.

This guidance document represents the Agency’s posi�on on this topic and is intended

solely as guidance. It does not have the force and effect of law, does not create, or confer

any rights for or on any person, and does not bind the U.S. Department of Agriculture

(USDA) or the public. Language sugges�ng that this guidance is mandatory (e.g., “shall,”

“must,” “required,” or “requirement”) should not be construed as binding unless the

terms quote from a statutory or regulatory requirement. The informa�on this document

contains may be made available to the public. While this document provides guidance for

users outside APHIS, APHIS employees may not deviate from the direc�ons provided

herein without appropriate jus�fica�on and supervisory concurrence.

2. Document Status

A. Review date: 8/15/2026. 

B. This document replaces VS Guidance 13407.2. 

3. Reason for Reissuance

VS is reissuing this guidance to update forma�ng, revise VS tes�ng procedures for

glanders, and add or amend several defini�ons to provide clarity to importers regarding

animal classifica�on and non-nega�ve test results.

4. Authority and References

A. Authori�es (Code of Federal Regulations (CFR)):

7 CFR 371.4 

9 CFR 93.304 

9 CFR 93.306 

9 CFR 93.308 

9 CFR 93.317 

9 CFR 93.324 

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

B. References:

World Organisa�on for Animal Health (WOAH) Manual of Diagnos�c Tests and Vaccines

for Terrestrial Animals: Chapters 3. 6. 3 Dourine; 3. 6.11 Glanders and melioidosis; 3. 

6. 6 Equine infec�ous anemia; and 3. 6. 8 Equine piroplasmosis.

VS Guidance 7000, Surveillance, Collec�on, and Submission of Suspected Exo�c Ticks

VS Guidance 13424, Procedures for the Import of Equines into the United States and

Approved Quaran�ne Facili�es

C. Definitions:

1) An�complementary (AC): Non-specific consump�on of complement;

indeterminate reac�on that occurs in the absence of an�gen. The causes of 

an�-complementary results are unknown, but a fas�ng blood sample may

improve the result. VS personnel should always submit clear serum without

evidence of hemolysis for complement fixa�on tests.

2) Cohorts: Contact animals from the same shipment. Cohorts vary by disease. The

specific defini�on of a cohort is described under each disease in Sec�on 6. B.

3) Classifica�on: The determina�on of an animal’s regulatory health status as

characterized by official diagnos�c tes�ng, including confirmatory or

supplemental tests when necessary. Test results will classify an animal as

nega�ve, suspect, or posi�ve for each disease. An animal must be classified

nega�ve to be considered eligible for release from quaran�ne.

4) Confirmatory test: Test method of high diagnos�c specificity used to confirm

results, usually posi�ve results, derived from other test methods, and thereby

determine the animal’s classifica�on. Horses determined to be nega�ve on

confirmatory tes�ng will be classified nega�ve for the target disease and may 

be eligible for release.

5) Non-nega�ve test result: A test result reported as posi�ve, inconclusive, or

suspect. Test results establish the regulatory classifica�on of an animal and are 

used to determine eligibility for release from quaran�ne.

6) Supplemental tests: Diagnos�c tests beyond the primary official test.

Supplemental tests are only performed on AC samples at the discre�on of VS

personnel.

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

5. Audience

VS employees, other Federal and State agencies, and members of the public.

6. Guidance

A. General

1) All imported horses, regardless of age, are tested for diseases as specified in 9 CFR 

93.308 using the following official tests:

a. Complement fixa�on test (CFT) for EP and dourine.

b. Agar gel immunodiffusion (AGID) test for EIA. 

c. Compe��ve enzyme-linked immunosorbent assay (cELISA) for EP. 

d. Double an�gen enzyme-linked immunosorbent assay (daELISA) for glanders.

2) Excep�ons to the general tes�ng requirements for imported horses:

a. Horses origina�ng and arriving directly from Iceland are exempt from tes�ng

for EP, dourine, glanders, and EIA.

b. Horses origina�ng and arriving directly from Australia and New Zealand are

exempt from tes�ng for dourine and glanders.

c. Horses origina�ng and arriving directly from Canada are exempt from tes�ng

for dourine, glanders, and EP. Canadian animal health officials test horses with

either the AGID test or the ELISA for EIA before the horses enter the United

States.

3) Government officials from the expor�ng country must cer�fy in wri�ng the iden�ty

of the origina�ng premises for all horses. This informa�on must appear on each

animal’s endorsed export cer�ficate or on addi�onal cer�fica�on the officials

provide to VS.

4) Unless otherwise specified, VS considers all horses transported in the same 

conveyance arriving at the U.S. port of entry one shipment for quaran�ne, tes�ng,

and release. Any non-nega�ve test results for an individual animal may affect the

release of the cohorts in the shipment. VS may consider any addi�onal

epidemiological informa�on presented in addi�on to import tes�ng to determine

the import status of all horses in the shipment.

5) During the horse’s first twenty-four (24) hours in quaran�ne, submiters (VS

personnel at the port) collect blood samples, process, package, and ship blood

samples to the VS Na�onal Veterinary Services Laboratories (NVSL) in Ames, IA in

accordance with port Standard Opera�ng Procedures.

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

6) If a horse tests non-nega�ve on the ini�al blood collec�on, handlers isolate it from 

the other horses in the shipment and may use addi�onal personal protec�ve

equipment based on the disease of concern and the presence or absence of clinical 

signs, in accordance with port Standard Opera�ng Procedures.

7) The veterinary medical officer (VMO)/receiving officer at the port examines the

animal and draws the samples; they can, based on test results or physical

examina�on, extend the quaran�ne period or refuse entry as prescribed in 9 CFR 

93.308(a)(4). The importer of record must pay for any extended quaran�ne period

and such extension is subject to the facility’s ability to keep horses for extended

periods.

8) Horses must be classified as nega�ve for dourine, glanders, EP, and EIA, based on

tes�ng conducted by NVSL, to be considered eligible for entry into the United

States. Horses refused entry must be either exported out of the United States

within ten (10) days of refusal no�fica�on or humanely euthanized.

9) When the results of the official dourine and EP CFT are an�complementary, the

laboratory performs supplemental tests, such as an indirect fluorescent an�body

(IFA) test. If a horse has yielded an�complementary results on previous tes�ng, the

responsible individual may no�fy the laboratory for awareness. VS personnel may 

use other VS-approved assays as supplemental tests. VS has no supplemental test

for EIA or glanders.

10) VS personnel must perform confirmatory tes�ng using the Western blot on all

samples that test posi�ve on the EP cELISA for Babesia caballi. 

11) VS personnel must perform confirmatory tes�ng using the Western blot on all

samples that test posi�ve on the daELISA for glanders.

12) The quaran�ne sta�on VMO releases NVSL test results to importers, agents, or

brokers. NVSL does not release any test results directly to importers, agents, or

brokers, except when given specific permission by the VMO at the quaran�ne

sta�on on a case-by-case basis.

13) NVSL advises the port VMO, Strategy and Policy Live Animal Import and Export

Directors, and the Equine Import veterinary staff officer of non-negative quarantine

test results.

B. Specific Informa�on and Collec�on Procedures for Each Disease

1) Dourine

a. General information

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

Dourine is a venereal disease of horses transmited primarily by sexual contact 

and caused by the protozoan Trypanosoma equiperdum. The organism may be 

present in the genital secre�ons of both male and female horses. The

incuba�on period, severity, and dura�on of disease vary. Clinical signs can

include fever, edema of the genitals and mammary glands, cutaneous

erup�ons, incoordina�on, facial paralysis, anemia, and emacia�on. While the

disease is o�en fatal, subclinical cases and latent carrier states can occur.

Infec�ons with other trypanosomes (e.g., T. evansi) may cause cross-reac�ons

with T. equiperdum serological assays.

b. Testing

1. The CFT is the official test for dourine. Posi�ve CFT results are equal to or

greater than a 2+ reac�on at a 1:5 dilu�on. Suspect test results are 1+

reac�ons at a 1:5 dilu�on.

2. VS personnel should immediately redraw blood samples from any horses 

tes�ng posi�ve or suspect and follow procedures outlined in sec�on 6.C.

3. When CFT results are an�complementary, NVSL performs supplemental 

tests on the sample obtained from the ini�al blood collec�on. If

supplemental tests are posi�ve or suspect, the port VMO should redraw 

blood samples immediately, submit to NVSL, and follow procedures

outlined in sec�on 6.C. NVSL performs official and supplemental tests on

the redrawn sample.

4. Refer to Figure 1. 

c. Cohorts

1. Intact animals over seven hundred and thirty-one (731) days, as well as any 

younger animals which have been bred, that were resident on the same

premises to the horses tes�ng posi�ve during the sixty (60) days

immediately prior to export are part of the cohort.

2. VS personnel will release castrated and immature equids if the animals had

nega�ve test results on the ini�al serum collec�on.

3. VS personnel hold cohorts for fourteen (14) days a�er the ini�al blood

collec�on, then rebleed and retest all cohorts. VS personnel may release

cohorts if all retest results are nega�ve. Cohorts that test posi�ve must

follow the procedures outlined in sec�on 6.C.

2) Glanders

a. General information

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

Glanders is a highly contagious bacterial disease caused by Burkholderia mallei. 

Infected animals may have nodules, abscesses, and ulcers in the respiratory

tract and skin. The average incuba�on period is two (2) weeks but varies from 

two (2) days to several months, and the disease may persist as chronic

infec�ons in inapparent carriers. In addi�on to affec�ng horses, glanders poses 

a significant public health risk. Humans can contract glanders through direct

contact with diseased animals or contaminated materials, with up to a ninetyfive (95) percent mortality rate in untreated cases. Infec�ons with other

bacteria (e.g., B. pseudomallei) may cause cross-reac�ons with B. mallei

serological assays.

b. Testing

1. The daELISA is the official test for glanders. Results on the daELISA are only

posi�ve or nega�ve. There is no suspect result. Posi�ve ELISA results are

equal to or greater than seventy (70) S/P percent. S/P is an op�cal density

(OD) measurement, here referring to the ra�o of the sample OD (S) to the

posi�ve control OD (P). 

2. If the results of the ini�al daELISA results are posi�ve, then the Western blot

will be used as a confirmatory test. Horsesthat are found nega�ve using the

Western blot confirmatory testwill be classified as nega�ve for glanders and

may be released. The final interpreta�on, including Western blot 

confirmatory tes�ng, will be included on the final NVSL report.

3. VS personnel should immediately redraw blood samples from any horses

tes�ng non-nega�ve using the daELISA and Western blot and follow the

procedures outlined in sec�on 6.C.

4. Refer to Figure 1. 

c. Cohorts

1. All animals in the shipment are cohorts.

2. VS personnel hold cohorts for fourteen (14) days a�er the ini�al blood

collec�on, then rebleed and retest all cohorts. VS personnel may release

cohorts if all retest results are nega�ve. Cohorts that test posi�ve must

follow the procedures outlined in sec�on 6.C.

3) Equine piroplasmosis (EP)

a. General information

1. EP, or babesiosis, is a �ck-borne protozoan disease of horses caused by

Babesia caballi or Theileria (Babesia) equi. Co-infec�ons with both

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

protozoan species may occur. Infec�ons may occur from �ck bites or

through contaminated needles or transfusions (iatrogenic infec�on). The

protozoan develops within the red blood cells of the host and within various

cells of competent �ck vectors. Tick-borne piroplasmosis infec�ons have a

one- (1-) to three- (3-) week incuba�on period; iatrogenic piroplasmosis

infec�ons may have a prolonged incuba�on period.

2. Clinical signs are variable and o�en nonspecific, but may include pale,

icteric, or hemorrhagic mucous membranes; fever; anemia; depression;

weakness; edema; and hemoglobinuria. Infected animals may remain

asymptoma�c carriers of these protozoan parasites for several years or

more.

b. Testing

1. The official tests for EP are the cELISA and CFT. The CFT is more likely to 

detect early infec�ons but may be nega�ve when tes�ng chronic carriers.

The cELISA targets chronic asymptoma�c carriers but may be nega�ve

during early stages of infec�on.

2. Results on the cELISA are only posi�ve or nega�ve. There is no suspect

result. Samples are considered posi�ve on cELISA if they exhibit a forty (40)

percent inhibi�on or higher result for either organism that causes EP.

3. Results on the CFT are only posi�ve or nega�ve. There is no suspect result. 

Posi�ve EP CFT results are equal to or greater than a 2+ reac�on at a 1:5

serum dilu�on.

4. Horses must be nega�ve on both cELISA and CFT to be considered nega�ve

for EP.

5. When CFT results are an�complementary, NVSL performs supplemental 

tests on the sample obtained from the ini�al blood collec�on. If

supplemental tests are non-nega�ve, the port VMO should immediately 

redraw and follow procedures outlined in sec�on 6.C.

6. Horses that test posi�ve for B. caballi by cELISA, but which are found

nega�ve using the Western blot confirmatory test, will be classified as

nega�ve for B. caballi and may be eligible for release. The final

interpreta�on, including Western blot confirmatory tes�ng, will be included

on the final NVSL report.

7. Refer to Figure 1. 

c. Cohorts

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

1. Horses resident on the same premises within the sixty (60) days

immediately prior to export are cohortsto the horsestes�ng posi�ve on the

cELISA or CFT.

2. VS considers addi�onal epidemiological informa�on (such as the presence

of �cks or the use of blood-contaminated medical equipment by personnel

accompanying the horses on the aircra�) when determining cohorts.

3. VS personnel hold cohorts for fourteen (14) days a�er the ini�al blood

collec�on, then rebleed and retest all cohorts using the cELISA and CFT for

B. caballi and/or T. equi as appropriate. VS personnel may release cohorts

if all retest results are nega�ve. Cohorts that test posi�ve must follow the

procedures outlined in sec�on 6.C.

d. Tick-infested horses

1. VS thoroughly inspects and treats all horses with an approved ectoparasite

spray as outlined in VS Guidance 13424.

2. Horses found to have atached �cks when examined will be held for

fourteen (14) days in quaran�ne and re-tested for piroplasmosis or may be 

refused entry.

3. VS personnel must submit the �cks collected to NVSL as outlined VS 

Guidance 7000. 

4) Equine infec�ous anemia (EIA)

a. General information

EIA is an acute or chronic viral disease of horses characterized by intermitent

fever, depression, weakness, weight loss, edema, and anemia. EIA is

transmited through blood from an infected animal by contaminated needles,

bloodsucking flies, or other blood contact. The incuba�on period usually ranges

from one (1) to three (3) weeks but can be as long as three (3) months.

b. Testing

1. The official test for EIA is the AGID (Coggins) test. Results of the AGID are

only posi�ve or nega�ve. There is no suspect result. If the EIA AGID test is

posi�ve on ini�al screening, NVSL performs the test again in duplicate on

the sample obtained from the first blood collec�on.

2. The port VMO should immediately redraw and resubmit blood samples to 

NVSL for any horses tes�ng posi�ve on the ini�al AGID test. If immediate

rebleed samples are posi�ve, the horse will be refused entry. VS should

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

immediately resample horses that test nega�ve on redrawn samples and

submit the samples to NVSL for tes�ng. VS will only release horses that are 

nega�ve to two sequen�al subsequent tests a�er an ini�al posi�ve test.

3. Refer to Figure 2. 

c. Cohorts

1. All animals in the shipment are cohorts.

2. VS refuses entry to cohorts without retes�ng and they are returned to the

country of origin; they cannot stay in import quaran�ne. The importer of

record may present cohorts for entry retes�ng a�er forty-five (45) days of

isola�on from EIA-posi�ve horses a�er VS reviews further epidemiological

information.

C. Procedures for Horses with Non-Nega�ve Results on Ini�al Sample Tes�ng for 

Glanders, Dourine, and EP

1) VS personnel should immediately redraw blood samples from any horses tes�ng

non-nega�ve for dourine, glanders, or EP and submit the samples to NVSL. The

importer of record can withdraw importa�on at any �me. In this case, tes�ng of

the cohorts may proceed as described for each disease (Sec�ons 6.B.1 – 6.B.3).

a. If nega�ve test results are received on the redrawn sample, the horse will be

allowed entry into the United States.

b. If non-nega�ve test results are received, the importer of record, on behalf of 

the owner, may choose to withdraw importa�on or con�nue to pursue

importa�on as described below.

2) Horses tes�ng non-nega�ve on the immediate rebleed may be held for fourteen

(14) days from the ini�al blood collec�on, which serves as day zero (0). On day 

fourteen (14), horses must be retested for the disease of concern.

a. If nega�ve test results are received on the day fourteen (14) test sample, the

horse is eligible for entry into the United States.

b. If non-nega�ve test results are received, and the importer of record con�nues

to pursue importa�on, the horse may be held for an addi�onal fourteen (14) 

days and retested.

3) Horses tes�ng non-nega�ve on day fourteen (14) may be held for an addi�onal 

fourteen- (14-) day period and must be retested for the disease of concern.

a. If nega�ve test results are received on the day twenty-eight (28) test sample,

the horse will be allowed entry into the United States.

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

b. If non-nega�ve test results are received, the horse will be refused entry with

no op�on for further tes�ng.

4) Refer to Figure 1. 

7. Inquiries

Please send all inquiries to:

Strategy and Policy, Live Animal Imports

301-851-3300, select op�on 2

Email: LAIE@usda.gov. 

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

Figure 1: Tes�ng of Imported Equines for Glanders, Dourine and Piroplasmosis

∗ If non-nega�ve a�er immediate rebleed, importer of record has discre�on to withdraw importa�on with

resul�ng refused entry or to hold for 14-day retest, provided that the facility can accommodate the request. 

∗ If importer of record requests to hold for 14-day intervals, the date of ini�al blood collec�on (or day 14 

rebleed) serves as day zero. Horses remaining non-nega�ve a�er 28 days will be refused entry.

∗ VS will refuse the en�re shipment if a glanders cohort horse tests posi�ve on the 14-day rebleed. 

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U.S. DEPARTMENT OF AGRICULTURE 

Marke�ng and Regulatory Programs

Animal and Plant Health Inspec�on Service 

Veterinary Services

VS Guidance 13407.3 

Figure 2: Tes�ng of Imported Equines for Equine Infec�ous Anemia

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